A Glossary for Chemical Approaches towards Unlocking the Trove of Metabolic Treasures in Actinomycetes

Actinobacterial natural products showed a critical basis for the discovery of new antibiotics as well as other lead secondary metabolites. Varied environmental and physiological signals touch the antibiotic machinery that faced a serious decline in the last decades. The reason was exposed by genomic sequencing data, which revealed that Actinomycetes harbor a large portion of silent biosynthetic gene clusters in their genomes that encrypt for secondary metabolites. These gene clusters are linked with a great reservoir of yet unknown molecules, and arranging them is considered a major challenge for biotechnology approaches. In the present paper, we discuss the recent strategies that have been taken to augment the yield of secondary metabolites via awakening these cryptic genes in Actinomycetes with emphasis on chemical signaling molecules used to induce the antibiotics biosynthesis. The rationale, types, applications and mechanisms are discussed in detail, to reveal the productive path for the unearthing of new metabolites, covering the literature until the end of 2020.


Introduction
The phylum Actinobacteria is one of the most diverse phyla of the Gram-positive bacteria, from which Actinomycetes are widely distributed in different natural habitats, including soils, lakes and oceans. Different Actinomycetes are also found in nature associated to many invertebrates and plants [1,2]. From the "antibiotic golden era", the period from 1940 to 1960 of antibiotic discovery by the phenotypic screening of soil microorganisms, Actinomycetes have been recognized as fruitful sources of diverse secondary metabolites (SMs) [3,4]. A considerable number of bioactive natural products have been detected from microbial origin, in which micro-organisms have assumed a potential role in strengthening the field of natural product discovery in the last decades [5]. Although products from a natural origin have a proven potency for the eradication of infectious diseases [6], antimicrobial resistance has seriously raised and has been recognized as a threat to humankind, with the prediction of~10 million deaths by 2050, if no action is applied [7]. Even with the average of 1000 new natural products, that have been discovered each year over the last two decades, the frequent rediscovery of already known compounds continues to be a key bottleneck in the field [8]. Additionally, recent advancements in the genome sequencing of Actinomycetes have shown "an innovation gap" between biosynthetic potential and isolated bioactive SMs [9,10]. The genomes of these Actinomycetes generally harbor many biosynthetic gene clusters (BGC) that encode paths for the creation of SMs, but with a plethora of silent genes of yet unobserved molecules [11]. The genome analysis revealed

Antibiotics at Sub-Inhibitory Concentrations
Transcriptomic studies have revealed that sub-inhibitory concentrations (SICs) of antibiotics can stimulate many regulators, especially those belonging to the PAS-LuxR family [22], which are classified under the quorum sensing regulators [23]. These PAS-LuxR family members comprise of PimM, NysRIV and PteF regulators of pimaricin, nystatin and filipin biosynthesis, respectively [24]. As reported, SICs as low as 1% of minimum inhibitory concentration (MIC) values, can trigger many phenotypic changes, such as the induction of biofilm formation, creating a virulence factor, increasing bacterial motility or activating the cryptic natural products biosynthesis [11]. In a marine Streptomyces spp., streptophenazine A (1) has been elicited with SICs of either bacitracin or tetracycline, to attain elevated titers by 2.2-fold at 0.1 μg/mL concentration [11]. Tetracycline also elicited streptophenazines E-H (5)(6)(7)(8), and enhanced streptophenazines A-D (1-4), while bacitracin induced streptophenazine H (8) with titers elevated between 2.6 and 10.7-fold [25]. The SIC of monensin induced cryptic isonitrile antibiotic SF2768 in the S. griseorubiginosus strain 574, while lincomycin (9) enhanced streptomycin (10) production in S. griseus, from 40-100 mg/mL [26] via binding to the ribosomes of Gram-positive bacteria and modifying the translational apparatus [27]. Additionally, lincomycin at one tenth of its MIC, enhanced the expression of the pathway-specific regulatory gene actII-ORF4, belonging to the SARP family, which is the main activator of ACT (11) biosynthesis in the enzyme gene cluster [28]. Moreover, chloramphenicol triggered a 1.5-to 6-fold enhancement in the biosynthesis of actinomycin D4 (12), calcium dependent antibiotics (CDAs, 13) and piperidamycin (14) in Streptomyces sp. [29], all of which exhibited a wide range of antimicrobial activities [30,31]. Interestingly, actinomycin D triggered the extrinsic pathway of apoptosis and acted synergistically with RG7787 to cause prominent tumor regression in mice [32]. It has been clinically tested for Wilms tumor of the kidney, rhabdomyosarcoma and trophoblastic tumors as well as down-regulating the expression of the stem cell transcription factor, Sox-2; a breast cancer fighter [33]. More interestingly, the SIC of actinomycin D specifically stimulated a p53-dependant transcription factor, which enhanced its killing power of p53 human tumor cells [34], and also prevented the Coxsackie B3 virus and HIV-

Antibiotics at Sub-Inhibitory Concentrations
Transcriptomic studies have revealed that sub-inhibitory concentrations (SICs) of antibiotics can stimulate many regulators, especially those belonging to the PAS-LuxR family [22], which are classified under the quorum sensing regulators [23]. These PAS-LuxR family members comprise of PimM, NysRIV and PteF regulators of pimaricin, nystatin and filipin biosynthesis, respectively [24]. As reported, SICs as low as 1% of minimum inhibitory concentration (MIC) values, can trigger many phenotypic changes, such as the induction of biofilm formation, creating a virulence factor, increasing bacterial motility or activating the cryptic natural products biosynthesis [11]. In a marine Streptomyces spp., streptophenazine A (1) has been elicited with SICs of either bacitracin or tetracycline, to attain elevated titers by 2.2-fold at 0.1 µg/mL concentration [11]. Tetracycline also elicited streptophenazines E-H (5)(6)(7)(8), and enhanced streptophenazines A-D (1-4), while bacitracin induced streptophenazine H (8) with titers elevated between 2.6 and 10.7-fold [25]. The SIC of monensin induced cryptic isonitrile antibiotic SF2768 in the S. griseorubiginosus strain 574, while lincomycin (9) enhanced streptomycin (10) production in S. griseus, from 40-100 mg/mL [26] via binding to the ribosomes of Gram-positive bacteria and modifying the translational apparatus [27]. Additionally, lincomycin at one tenth of its MIC, enhanced the expression of the pathway-specific regulatory gene actII-ORF4, belonging to the SARP family, which is the main activator of ACT (11) biosynthesis in the enzyme gene cluster [28]. Moreover, chloramphenicol triggered a 1.5-to 6-fold enhancement in the biosynthesis of actinomycin D4 (12), calcium dependent antibiotics (CDAs, 13) and piperidamycin (14) in Streptomyces sp. [29], all of which exhibited a wide range of antimicrobial activities [30,31]. Interestingly, actinomycin D triggered the extrinsic pathway of apoptosis and acted synergistically with RG7787 to cause prominent tumor regression in mice [32]. It has been clinically tested for Wilms tumor of the kidney, rhabdomyosarcoma and trophoblastic tumors as well as down-regulating the expression of the stem cell transcription factor, Sox-2; a breast cancer fighter [33]. More interestingly, the SIC of actinomycin D specifically stimulated a p53-dependant transcription factor, which enhanced its killing power of p53 human tumor cells [34], and also prevented the Coxsackie B3 virus and HIV-1 [12,13], making actinomycin D one of the most talented candidates for medicinal development purposes [35]. Furthermore, SICs of some ribosome-targeted antibiotics, such as thiosteptone 1 [12,13], making actinomycin D one of the most talented candidates for medicinal development purposes [35]. Furthermore, SICs of some ribosome-targeted antibiotics, such as thiosteptone and spectinomycin, can exert a 4-fold growth in undecylprodigiosin (RED, 15) production in S. coelicolor M145 [36], which attains a wide range of antimycotic, antiprotozoal and larvicidal activities ( Figure 2) [37].

Antibiotics as Autoregulators of Their Own Biosynthesis
Recent studies have focused on the role of atypical response regulators (ARRs) that are included in the antibiotic biosynthetic pathways, such as the cluster-situated regulator (CSR) activator JadR1, an OmpR-type ARR of S. venezuelae, included in the biosynthesis of jadomycin B (JdB, 16) [38]. Generally, antibiotics exert their regulatory effects via CSRs

Antibiotics as Autoregulators of Their Own Biosynthesis
Recent studies have focused on the role of atypical response regulators (ARRs) that are included in the antibiotic biosynthetic pathways, such as the cluster-situated regulator (CSR) activator JadR1, an OmpR-type ARR of S. venezuelae, included in the biosynthesis of jadomycin B (JdB, 16) [38]. Generally, antibiotics exert their regulatory effects via CSRs that are linked to BGCs or pleiotropic regulators (regulators that control the biosynthesis of multiple SMs) [3]. JdB was found to specifically interact with JadR1 and control its own production in S. venezuelae in a highly organized feedback regulatory mechanism [39]. The feedback regulatory mechanism is best expressed by JadR * , a TetR family regulator that responds to the JdB intermediates [2,3-dehydro-UWM6 (DHU) and dehydrorabelomycin (DHR)] [24]. This indicates that antibiotics and their biosynthetic intermediates have the ability to share a role as ARRs through the modulation of their own biosynthesis. Additionally, ChlF1, which is also a TetR family regulator included in chlorothricin (17) biosynthesis in S. antibioticus, was found to split from its target promoters in the occurrence of chlorothricin with its biosynthetic intermediates (demethyl salicyloyl chlorothricin and deschloro-chlorothricin), followed by the interaction with this regulator [40]. Moreover, CalR3, which is a TetR family regulator involved in the calcimycin (18) biosynthesis of S. chartreusis NRRL 3882, responds to calcimycin and its biosynthetic intermediates (dimethyl-salicycloyl chlorothricin and deschloro-chlorothricin) [40]. Furthermore, NosP regulates the production of nosiheptide (NOS) (19) in S. actuosus, in response to both nosiheptide as well as its biosynthetic intermediates [41]. Nosiheptide exhibited a strong anti-mycobacterial activity against M. avium and M. intracellulare, with obtained MICs in the range of 0.024 to 1.56 µg/mL [42]. Apart from Streptomyces, the Actinomycete Amycolatopsis mediterranei plays a role as an extracellular signaling molecule regulating rifamycin B (20) export in a feedback mechanism [41], while 2,4-diacetylphloroglucinol (2,4-DAPG, 21) and its biosynthetic intermediate monoacetylphloroglucinol can regulate the biosynthesis of 2,4-DAPG in Pseudomonas fluorescens ( Figure 3) [43]. that are linked to BGCs or pleiotropic regulators (regulators that control the biosynthesis of multiple SMs) [3]. JdB was found to specifically interact with JadR1 and control its own production in S. venezuelae in a highly organized feedback regulatory mechanism [39]. The feedback regulatory mechanism is best expressed by JadR * , a TetR family regulator that responds to the JdB intermediates [2,3-dehydro-UWM6 (DHU) and dehydrorabelomycin (DHR)] [24]. This indicates that antibiotics and their biosynthetic intermediates have the ability to share a role as ARRs through the modulation of their own biosynthesis. Additionally, ChlF1, which is also a TetR family regulator included in chlorothricin (17) biosynthesis in S. antibioticus, was found to split from its target promoters in the occurrence of chlorothricin with its biosynthetic intermediates (demethyl salicyloyl chlorothricin and deschloro-chlorothricin), followed by the interaction with this regulator [40]. Moreover, CalR3, which is a TetR family regulator involved in the calcimycin (18) biosynthesis of S. chartreusis NRRL 3882, responds to calcimycin and its biosynthetic intermediates (dimethyl-salicycloyl chlorothricin and deschloro-chlorothricin) [40]. Furthermore, NosP regulates the production of nosiheptide (NOS) (19) in S. actuosus, in response to both nosiheptide as well as its biosynthetic intermediates [41].

Antibiotics as Autoregulators of Other Biosynthetic Pathways
One of the important examples of this type of autoregulator is JadR2, a common TetR family regulator that modulates JdB biosynthesis in S. venezuelae via the direct repression of jadR1, which is sensitive to JdB end products [44]. Interestingly, JdB combines with apramycin, hygromycin B or kanamycin, then modulates the receptor DNA-binding activity of

Antibiotics as Autoregulators of Other Biosynthetic Pathways
One of the important examples of this type of autoregulator is JadR2, a common TetR family regulator that modulates JdB biosynthesis in S. venezuelae via the direct repression of jadR1, which is sensitive to JdB end products [44]. Interestingly, JdB combines with apramycin, hygromycin B or kanamycin, then modulates the receptor DNA-binding activity of AvaR2, leading to an enhancement of the production of the macrocyclic lactone avermectin (22) [3], which attains significant anthelmintic and insecticidal/acaricidal activities ( Figure 3) [45].

Antibiotics as Cross-Regulators of Other Antibiotics Biosyntheses
RedZ, a CSR of the red gene cluster, was found to modulate the production of RED, ACT as well as CDA in S. coelicolor [46]. The same idea was discussed in a study that reported that FscRI (the candicidin CSR) was detected to regulate the overall biosynthesis of both candicidin (23) and antimycin (24), the end products of a different BGC in S. albidoflavus S4 [47]. In the same context, JadR1 was observed to control JdB as well as chloramphenicol (25) production in S. venezuelae [39], while GdmRIII proved to enhance geldanamycin (26) and elaiophylin (27) in S. autolyticus CGMCC0516 [48]. It is worth mentioning that geldanamycin is a unique antibiotic exhibiting virucidal, ischemia protective and antineoplastic activities (Figure 4) [49]. AvaR2, leading to an enhancement of the production of the macrocyclic lactone avermectin (22) [3], which attains significant anthelmintic and insecticidal/acaricidal activities ( Figure 3) [45].

Antibiotics as Cross-Regulators of Other Antibiotics Biosyntheses
RedZ, a CSR of the red gene cluster, was found to modulate the production of RED, ACT as well as CDA in S. coelicolor [46]. The same idea was discussed in a study that reported that FscRI (the candicidin CSR) was detected to regulate the overall biosynthesis of both candicidin (23) and antimycin (24), the end products of a different BGC in S. albidoflavus S4 [47]. In the same context, JadR1 was observed to control JdB as well as chloramphenicol (25) production in S. venezuelae [39], while GdmRIII proved to enhance geldanamycin (26) and elaiophylin (27) in S. autolyticus CGMCC0516 [48]. It is worth mentioning that geldanamycin is a unique antibiotic exhibiting virucidal, ischemia protective and anti-neoplastic activities ( Figure 4) [49].

Antibiotic Remodeling Compounds (ARCs)
A family of fully synthetic chemicals with the ability to modify the fatty acid biosynthesis, known as antibiotic remodeling compounds (ARCs), have been recently investigated and observed to change fatty acid pools through countering the key biosynthetic enzymes as FabI [50], and displaying a superior current of acetyl-CoA for the antibiotic biosynthesis [50]. Notably, a large number (>30,000) of ARCs were screened to understand the regulatory network by using chemical perturbation of secondary metabolism [51]. Regarding the ability to remodel ACT production, the above mentioned screening yielded 19 compounds, of which ARC2, ARC3, ARC4 and ARC5 were the most active [52]. ARC2, as the top active, can encourage the buildup of elevated levels of unsaturated fatty acids in Streptomyces sp., which enhanced the production of erythromycin (28), oligomycin (29), monensin B (30) and ACT [53], and achieved a three-fold increase in germicidins A-C (31-33) [29]. Moreover, it enhanced the production of the siderophore, desferrioxamine B (34)

Antibiotic Remodeling Compounds (ARCs)
A family of fully synthetic chemicals with the ability to modify the fatty acid biosynthesis, known as antibiotic remodeling compounds (ARCs), have been recently investigated and observed to change fatty acid pools through countering the key biosynthetic enzymes as FabI [50], and displaying a superior current of acetyl-CoA for the antibiotic biosynthesis [50]. Notably, a large number (>30,000) of ARCs were screened to understand the regulatory network by using chemical perturbation of secondary metabolism [51]. Regarding the ability to remodel ACT production, the above mentioned screening yielded 19 compounds, of which ARC2, ARC3, ARC4 and ARC5 were the most active [52]. ARC2, as the top active, can encourage the buildup of elevated levels of unsaturated fatty acids in Streptomyces sp., which enhanced the production of erythromycin (28), oligomycin (29), monensin B (30) and ACT [53], and achieved a three-fold increase in germicidins A-C (31-33) [29]. Moreover, it enhanced the production of the siderophore, desferrioxamine B (34) and E (35) in S. pristinaespiralis, and the antibiotics doxorubicin (36) and baumycin (37) in S. peucetius [52], all of which attain powerful anticancer properties [52]. Interestingly, ARC2, ARC3, ARC4 and ARC5 are referred to as the "ARC2 series" for antibioticremodeling compounds, as they had similar structures and stimulated blue pigmentation during growth on a solid medium. They can reduce yields of the prodiginines and enhance the production of two other S. coelicolor secondary metabolites and germicidin~3-fold; whereas yields of the daptomycin-like calcium-dependent antibiotic (CDA) were reduced 2-fold [54]. Accordingly, the ARC2 series can therefore pleiotropically remodel secondary metabolism in S. coelicolor [54]. On the other hand, ARC6 elevated the levels of ACT in Streptomyces via the same mechanism, but with limitation to S. coelicolor, which indicates that it acts more as a species-specific synthetic signal [51]. Recently, a chlorinated analog of ARC2, named Cl-ARC, was employed to trigger the expression of CBGs in an unbiased screening regimen [52]. This resulted in the elicitation of three rare and valuable antibiotics: oxohygrolidin (38) (antifungal and insecticidal [55]) from S. ghanaensis ATCC 14672, 9methylstreptimidone (39) (anti-inflammatory, antiproliferative and antibacterial [56]) from S. hygroscopicus ATCC 53653 and dynactin (40), as well as the known antibiotics nonactin (41), monactin (42) and trinactin (43), from WAC0256 [57]. All of them, especially dynactin, showed motility impairing potency against P. viticola, Phytophthora capsici and Aphanomyces cochlioides zoospores, which indicates non-specific activities toward peronosporomyctes ( Figure 5) [58].

Histone Deacetylase Inhibitors (HDACIs)
HDACs are members of a large family of epigenetic drugs that affect histone deacetylation, cause apparent changes in the chromatin structure and attain approval for treating serious cases, such as lymphoma and myeloma [47]. They catalyze many other deacylation reactions, such as demalonylation, decrotonylation or desuccinylation, besides possessing many critical enzyme-independent functions [59,60]. They are classified into four classes: class I (HDAC1, 2, 3 and 8); class II [further sub-grouped into class IIa (HDAC4, 5, 7 and 9) as well as class IIb (HDAC6 and 10)]; class III (HDAC11) and class IV (SIRT1-7) [61]. On the other hand, histone deacetylase inhibitors (HDIs) block these HDAC enzyme activities and inhibit their deacetylation reaction. They bind to the zinc ion inside the catalytic sites of the enzymes, leading to the obstruction of the substrate admission to these sites, thus blocking their action. They include hydroxymates, such as suberoylanilide hydroxamic acid (vorinostat), LBH589 (panobinostat), TSA (trichostatin A) and PXD101 (belinostat) [62,63]. Short-chain fatty acids, such as sodium butyrate (SB) and valproic acid (VPA), prohibit class I and IIa HDACs, while benzamides, such as MS275 (entinostat), and depsipeptides, such as FK228 (romidepsin), prohibit some of the class I HDACs [62,63].
Their main mechanism of activating BGCs is directed towards three HDAC-like genes, which transcriptionally control the biosynthetic pathways in several Streptomyces strains, resulting in the modification of the nucleoid structure [64] and enhancement of antibiotic production [18]. SB and VPA (25 mM) have the ability to elicit ACT, as well as five other cryptic pathways with deoxysugar synthetase, hopanoids, sesquiterpene cyclase, germicidin and coelibactin in S. coelicolor A3 strain M145 [64].

Hormone-like Signaling Molecules (Autoregulators)
Hormone-like signaling molecules are low molecular weight compounds that can enhance antibiotic production, and/or induce many morphological differentiations at nanomolar concentrations [65]. The first and most common identified autoregulator is the A-factor that greatly induces streptomycin production in S. griseus [66]. In general, autoregulators enhance SMs production via passive diffusion into the cells, accumulation beyond the critical concentration [15] as well as targeting the regulatory genes for "clustersituated regulators" (CSRs), which are linked to antibiotic BGCs [67]. By identifying a total of 24 autoregulators from more than 12 Streptomyces species, they can be categorized into 5 major categories: γ-butyrolactones (GBLs), aromatic furans (AFs), γ-butenolides, PI factor and N-methylphenylalanyl-dehydrobutyrine diketopiperazine (MDD) [21].

Aromatic Furans
The first and more common identified aromatic furan was hydroxymethylfuran, which enhanced the methylenomycin genes in S. coelicolor for the production of methylenomycin A [74]. The second aromatic furan was 7ae, an enzyme inhibitor of phosphopantetheinyl transferase, which is included in the activation of the acyl carrier protein of fatty acid biosynthesis, resulting in the production of ACT in cultures of S. coelicolor [79].

Metabolic Signals: GlcNAc and Siderophores
Metabolic signals are prevalent minute molecules controlling antibiotic biosynthesis and morphological differentiation in Streptomyces sp., when directly applied to cultures, and are considered part of primary metabolism [90]. The most common one is N-acetylglucosamine, that is released from the cell wall during autolysis by the action of chitinases, and is considered as a major food source for Streptomycetes Spp. Some other signaling molecules that are still poorly understood are strain-specific peptides (such as the small peptide goadsporin) as well as the protein factor C [67], in addition to glutamate, which is favored over GlcNAc by S. coelicolor [91]. It has been reported that metabolic signals act via a signaling cascade, from an extracellular signaling nutrient towards the enhancement of antibiotic production [21]. A related study investigated the effect of GlcNAc on the metabolic profiles of nine sponge-derived Actinomycetes, from which only Micromonospora sp. RV43, Rhodococcus sp. RV157 and Actinokineospora sp. EG49 revealed changes in their metabolic profiles [92]. The elicitor induced the bioformation of 3-formylindole (60) and guaymasol (61) in Micromonospora sp. RV43, bacillibactin (62) and surfactin lipopeptide 17 (63) in Rhodococcus sp. RV157 as well as the poorly expressed new metabolites, Actinosporins E-H (64)(65)(66)(67) in Actinokineospora sp. EG49 [93]. These results highpoint the use of NMR fingerprinting to perceive fluctuations in metabolic profiles, following the addition of the pleiotropic regulator, GlcNAc, which suppresses metabolites, induces new metabolites and also increases the production of minor compounds [92]. Recently, the HR-ESI-MS technique was applied for dereplication studies, in which G-2N (68) and saptomycin F (69) were dereplicated, while the antitrypanosomal new compounds fridamycin H (70) fridamycin I (71) and Actinosporin C, D, and G (72-75) were isolated from the solid culture of the sponge-associated Actinomycete, Actinokineospora spheciospongiae sp. nov. [72]. Moreover, five kigamicin derivatives (76)(77)(78)(79)(80) were also elicited from the fermentation extract of Amycolatopsis alba DSM44262∆abm9, after the introduction of 25 mM GlcNAc [94]. From them, Kigamicin D was highlighted as a powerful antitumor that inhibited the growth of various mouse tumor cell lines at IC 50 of about 1 µg/mL [95].
Interestingly, desferrioxamine is a fungal growth factor and one of the most common siderophores among terrestrial Actinomycetes, and the original supplier of the amount of iron available to the receiving strain included in the binary interaction [96], which indorses developmental features, such as sporulation and SMs production. Desferrioxamine E stimulated the growth, cell differentiation and enhancement of streptomycin, neomycin (81) and kanamycin (82) production (Figure 7) in S. tanashiensis [96]. Interestingly, the S. coelicolor mutant defective in desferrioxamine biosynthesis (KY1), showed impaired growth and development on Bennett's/glucose solid medium, an impairment that is probably due to an iron deficiency and confirmed the previous findings [97]. The KY1 suggested that the bacteria have other ferric uptake system(s), combined with speculating that another peptidic siderophore, that are termed coelichelin and existed in S. coelicolor A3(2) [98]. Both S. coelicolor and S. tanashiensis were found to grow and develop well on Bennett's medium supplied with maltose, which further suggested that the siderophore production in Streptomyces strains is under complex regulation that links to carbohydrates in addition to ferric limitation [96].

Rare Earth Elements (REEs)
One of the most interesting eliciting groups involved in the expression of silent BGCs is REEs, which have recently been implicated for the production of diverse SMs, including antibiotics, pigments, mycotoxins and phytotoxins [64]. Since REEs are distributed far and wide through the world, microorganisms may have assimilated the ability to respond to minute levels of these elements as an "abiotic" stress over their long evolutionary history, probably as a means of adapting to predominant conditions [99]. Consequently, ACT, dactinomycin (83), actinomycin, streptomycin and bacilysin (84) production by S. coelicolor, S. antibioticus, S. parvulus, S. griseus and B. subtilis 168, respectively, has been found to be enhanced, upon the addition of scandium (Sc 3+ ) [15]. Scandium exerted its eliciting action at the level of transcription of positive pathway-specific regulatory genes, as clarified by the apparent up-regulation of actII-ORF4 applied in S. coelicolor cells, with the critical importance of the bacterial alarmone, guanosine-5 -diphosphate 3 -diphosphate, for ACT overproduction [100]. Additionally, it is thought to act via enhancing enzyme production (α-amylase and bacilysin at the transcriptional level) and secondary metabolism, which was also observed upon adding Sc to B. subtilis [101]. Recently, a relevant study showed that cerium (Ce), europium (Eu) and yttrium (Y) upregulated the actII-ORF transcripts, resulting in the alteration of the transcription of 17 genes fitting to the same number of SMs biosynthetic gene clusters in S. coelicolor [15]. Recently, lanthanum chloride (LaCl 3 ) was tested in 50 Actinobacterial strains for their anti-microbial activities against a variety of pathogens, including C. albicans, Clostridium difficile, S. aureus, MRSA and Pseudomonas aeruginosa. Fifteen strains attained enhanced antimicrobial properties upon adding LaCl 3 , with a remarkable enhancement of antimycin-type compounds in strain R818, especially the antifungal compound, urauchimycin D (85) (Figure 8). In the same context, lanthanum chloride (50 µM) can provoke antibacterial biosynthetic pathways, upon being added to Promicromonospora kermanensis DSM 45485, resulting in the production of the antimycin-type compounds [64]. The main benefit of using REEs in culture media for augmenting antibiotic production, is the absence of need for preceding knowledge of genetic engineering on the strains examined. Since REEs are distributed ubiquitously throughout the world, it is possible that microorganisms have acquired the ability to respond to minute levels of such elements over the course of their evolutionary history, probably as a way of adapting their physiology to the prevailing conditions [102].

Organic Solvents
Ethanol and DMSO have also been used to elicit the biosynthesis of microbial compounds, possibly by the mistranslation or induction of the stress response [106]. Upon addition of DMSO, a three-fold increase was obtained in chloramphenicol and tetracenomycin C (88) production by both S. venezuelae ATCC 10712 and S. glaucescens, as well as a remarkable expression of the antibiotic coding BGCs in Promicromonospora kermanensis [107]. Likewise, a two-fold enhancement of thiostrepton (89) and a three-fold increase in both chloramphenicol and tetracenomycin C, respectively, were also observed in S. azureus ATCC 14921 upon supplementation with 3% (v/v) DMSO [107]. Moreover, DMSO has been shown to trigger prodigiosin (90) production by S. lividans and alter the antibiotic

Organic Solvents
Ethanol and DMSO have also been used to elicit the biosynthesis of microbial compounds, possibly by the mistranslation or induction of the stress response [106]. Upon addition of DMSO, a three-fold increase was obtained in chloramphenicol and tetracenomycin C (88) production by both S. venezuelae ATCC 10712 and S. glaucescens, as well as a remarkable expression of the antibiotic coding BGCs in Promicromonospora kermanensis [107]. Likewise, a two-fold enhancement of thiostrepton (89) and a three-fold increase in both chloramphenicol and tetracenomycin C, respectively, were also observed in S. azureus ATCC 14921 upon supplementation with 3% (v/v) DMSO [107]. Moreover, DMSO has been shown to trigger prodigiosin (90) production by S. lividans and alter the antibiotic profiles of Bacillus circulans and B. polymyxa, which was detected by LC-MS analysis [107]. Furthermore, Promicromonospora kermanensis DSM 45485 showed noteworthy anti-MRSA activity, after inoculation in an ISP2 broth medium containing both DMSO and VPA, which triggered a modification of the chromatin structure and an activation of ACT BGCs [64]. Adding ethanol to S. venezuelae ISP5230 in a D-galactose-L-isoleucine production medium, elicited the production of JdB via the induction of a heat shock response in S. venezuelae ISP5230, which served as a metabolite precursor, or alteration of membrane permeability [108] ( Figure 8).

HITES Approach
A recent approach has been designated for the unearthing of small molecule elicitors of silent BGCs, named the chemogenetic high-throughput screening approach, "HiTES", which has been formerly and successfully applied to the Gram-negative bacterium, Burkholderia thailandensis. In HiTES, a reporter gene is presented into a silent BGC of interest to deliver a rapid read-out for expression which utilizes a library of diverse SMs, and the effect of each compound from that library is assayed on the expression of the reporter gene. This is followed by inspecting the detected elicitors from the library, to identify both the production and the induction mechanism of the silent BGC [52,109]. Engaging this approach, activating BGCs can be applicated in a targeted fashion within genetically tractable strains, by changing the elicitors or their concentrations or both. Thus, the level of activation can be tuned and elevated up to~150-fold, resulting in a significant enhancement of SMs biosynthesis from a given silent BGC. [109]. In a relevant study, six potential antibiotic elicitors were identified through the screening of a commercially-available natural products library (ca. 500 compounds) by HiTES. The most remarkable ones included pleiotropic elicitors, such as ivermectin b1a and etoposide, which attained the maximum surugamide BGC elicitation and changed the SMs profile of S. albus J1074-14 [110]. Accordingly, a number of novel SMs were elicited, isolated and characterized, involving surugamides A (91), D (92), F (93), F2 (94), F3 (95), G-J (96-99), acyl-surugamide A (100) [111], albucyclones A-F (101-106), albuquinone A (107), mansouramycin analog (108) and mansouramycin C (109) [112], all of which exerted significant cytotoxic activities [113,114]. Acyl-surugamide A exhibited a significant antifungal activity against Saccharomyces cerevisiae with an IC 50 of 3.5 µM, while the mansouramycin analog had a broad anticancer activity, with IC values ranging from 0.250 µM against 36 diverse cancer cell lines [112]. It synergized with sorafenib, a strong inducer of the mitochondrial permeability transition and a talented anticancer drug candidate to induce cell death in A549 cells [115]. These results underline the value of HiTES in the production of bioactive SMs, as was seen with the activation of the silent BGC in S. albus, which harbors an excess of silent BGCs, strain J1074 [116], called SurA, that produces surugamides as a result [117]. Investigating the mode of SurA induction led to discovery of a pathway-specific repressor, SurR, which is a pathway-specific transcriptional regulator of the GntR family that silences the Sur cluster, all of which explain why little or no products are observed under standard laboratory conditions. Ivermectin b1a and etoposide elicitors induced a 2-2.5-fold downregulation of surR expression, resulting in their stimulatory activities [118]. A study has successfully combined bioactivity assays with (HiTES), to access cryptic, bioactive metabolites in Saccharopolyspora cebuensis, with the suppression of E. coli growth as a read-out. The results highlighted the identification of a novel lanthipeptide, cebulantin (110), elicited with a number of elicitors, including procaine, furosemide and fenofibrate, which showed selectivity against Gram-negative bacterial growth, notably that of diverse Vibrio pathogens [119]. HiTES has been fruitfully used to awake BGCs responsible for antibiotic production in S. hiroshimensis, via the employment of atenolol, which permits a selective inhibitory activity against E. coli and Acinetobacter baumannii [120]. Atenolol proved to be a pleiotropic global regulator that affected SM in S. hiroshimensis, and enhanced the production of taylorflavins A (111) and B (112), pyridindolol (113) 6,8-dihydroxy-3-methylisocoumarin (114), 6,7,8-trimethoxy-3-methylisocoumarin (115) and hiroshidine (116) [117]. Moreover, S. lavendulae, S. hiroshimensis and Amycolatopsis kerathiniphila, the producers of streptothricin, keratinimicin, and prodigiosin, respectively, can never produce an antibiotic under normal experimental conditions in an R4 growth medium, but, with the help of an elicitor, they can. In S. lavendulae and S. hiroshimensis, 7,2 -dimethoxyflavone, isobutylmethylxanthine and methoxyvone induced the synthesis of anti-B. subtilis metabolites, including the antimalarial primaquine (117) [121] and the local anaesthetic procaine (118) [122]. Similarly, aspartame-and tandutinib triggered the bioformation of baccatin III (119) and isoscopoletin (120) (Figure 9) in A. keratiniphila, which resulted in the enhancement of E. coli growth. Finally, the metabolomes that most enhanced E. coli growth in S. hiroshimensis, were elicited by aceclidine, glucosamine and dexchlorpheniramine [117]. Applying the same technique, goadsporin isolated from Streptomyces sp. TP-A0584 induced sporulation of many Streptomyces species and the production of RED in S. lividans [123].

Cumulative Effect of More than One Chemical Elicitor
Nine specific chemical elicitors were assessed for their potential on tacrolimus (121) accumulation in S. tsukubaensis, the low-yield drug that is clinically approved for prophylaxis against post-transplantation organ rejection ( Figure 10). Tacrolimus acts via inhibiting the proliferation of the T cells and the expression of interleukin-2 inside these cells, and it proved to be a potent immunosuppressive alternative to cyclosporine in liver transplantation cases [124]. The investigation results revealed that SB, DMSO and LaCl 3 can enhance tacrolimus accumulation by more than 30%, and combining DMSO and La increased the yield by 64.7% (303.60 mg/L), compared to the control. A total of 89 SMs, including sugars, amino acids, organic acids, flavonoids and fatty acids, have been detected with a different intracellular metabolism, which is highly correlated with La. Regarding La and SB treatments, diverse groups of resultant amino acids can be created, such as pyruvates (valine and leucine) and aspartates (aspartate, homoserine, lysine and isoleucine) from La treatment, as well as pyruvates (valine and alanine) and aromatic amino acids (tyrosine, phenylalanine and tryptophan) from SB treatment. To obtain an explanation, the impact of amino acids on the tacrolimus production has been studied, following the mechanism of cumulative action of DMSO and La. Different metabolic pathways have been enriched as TCA cycle, amino acid metabolism and benzoate degradation via CoA, compared to the single added chemical elicitor. Although the specific mechanism is still unclear, an obvious response characterization of intracellular metabolism can be detected in the main precursor pathways of tacrolimus synthesis. Additionally, the transition of the methylmalonyl-CoA synthetic pathway in the mutual applications of chemical elicitors has also provided an operative approach to recover tacrolimus, by minimizing the competition for pyruvate and/or acetyl-CoA in tacrolimus biosynthesis. It was clearly concluded that SB, DMSO and REEs (La, Sc, Ho) generated better stimulating effects on tacrolimus accumulation than other solitary chemical elicitors. In a brief conclusion, the augmented metabolic pathways (pentose phosphate and glycolysis pathways) can be greatly enhanced with DMSO treatment, such as methoxymalonyl-CoA, DHCHC, methylmalonyl-CoA, malonyl-CoA and allylmalonyl-CoA. On the other hand, SB treatment triggered PPP and the biosynthesis of unsaturated fatty acids, while the La treatment only enhanced the amino acid metabolism [125].

Cumulative Effect of More Than One Chemical Elicitor
Nine specific chemical elicitors were assessed for their potential on tacrolimus (121) accumulation in S. tsukubaensis, the low-yield drug that is clinically approved for prophylaxis against post-transplantation organ rejection ( Figure 10). Tacrolimus acts via inhibiting the proliferation of the T cells and the expression of interleukin-2 inside these cells, and it proved to be a potent immunosuppressive alternative to cyclosporine in liver transplantation cases [124]. The investigation results revealed that SB, DMSO and LaCl3 can enhance tacrolimus accumulation by more than 30%, and combining DMSO and La increased the yield by 64.7% (303.60 mg/L), compared to the control. A total of 89 SMs, including sugars, amino acids, organic acids, flavonoids and fatty acids, have been detected with a different intracellular metabolism, which is highly correlated with La. Regarding La and SB treatments, diverse groups of resultant amino acids can be created, such as pyruvates (valine and leucine) and aspartates (aspartate, homoserine, lysine and isoleucine) from La treatment, as well as pyruvates (valine and alanine) and aromatic amino acids (tyrosine, phenylalanine and tryptophan) from SB treatment. To obtain an explanation, the impact of amino acids on the tacrolimus production has been studied, following the mechanism of cumulative action of DMSO and La. Different metabolic pathways have been enriched as TCA cycle, amino acid metabolism and benzoate degradation via CoA, compared to the single added chemical elicitor. Although the specific mechanism is still unclear, an obvious response characterization of intracellular metabolism can be detected in the main precursor pathways of tacrolimus synthesis. Additionally, the transition of the methylmalonyl-CoA synthetic pathway in the mutual applications of chemical elicitors has also provided an operative approach to recover tacrolimus, by minimizing the competition for pyruvate and/or acetyl-CoA in tacrolimus biosynthesis. It was clearly concluded that SB, DMSO and REEs (La, Sc, Ho) generated better stimulating effects on tacrolimus accumulation than other solitary chemical elicitors. In a brief conclusion, the augmented metabolic pathways (pentose phosphate and glycolysis pathways) can be greatly enhanced with DMSO treatment, such as methoxymalonyl-CoA, DHCHC, methylmalonyl-CoA, malonyl-CoA and allylmalonyl-CoA. On the other hand, SB treatment triggered PPP and the biosynthesis of unsaturated fatty acids, while the La treatment only enhanced the amino acid metabolism [125].

Combinatorial Engineering Approach
A novel combinatorial engineering approach, depending on the identification of targets that are involved in both the metabolic and transcriptional regulation of many antibiotics, has been totally constructed. Ascomycin (FK520) (122) (Figure 10) is a macrocyclic antibiotic that attains antifungal and immunosuppressive potencies, but, unfortunately, it attains relatively low titer and yield, which hampered its pharmaceutical application. Therefore, a rich ascomycin-producing strain, such as S. hygroscopicus, was employed and screened employing DMSO, which apparently resulted in good production enhancement. The intracellular metabolic and transcriptional profiles were compared after the introduction of both DMSO and control, where the potential target genes involved in metabolic precursor pathways (zwf and aroA) and other pathways included in the transcriptional

Combinatorial Engineering Approach
A novel combinatorial engineering approach, depending on the identification of targets that are involved in both the metabolic and transcriptional regulation of many antibiotics, has been totally constructed. Ascomycin (FK520) (122) (Figure 10) is a macrocyclic antibiotic that attains antifungal and immunosuppressive potencies, but, unfortunately, it attains relatively low titer and yield, which hampered its pharmaceutical application. Therefore, a rich ascomycin-producing strain, such as S. hygroscopicus, was employed and screened employing DMSO, which apparently resulted in good production enhancement. The intracellular metabolic and transcriptional profiles were compared after the introduction of both DMSO and control, where the potential target genes involved in metabolic precursor pathways (zwf and aroA) and other pathways included in the transcriptional regulation (luxR, iclR, fadR and fkbN) have been detected. The peak yield of ascomycin (1258.30 ± 33.49 mg/L), 4.12-fold higher than the control yield (305.60 ± 16.90 mg/L), has resulted from the deviation of carbon flux towards ascomycin accumulation, leading to both metabolic and transcriptional regulation. Combinatory analysis revealed that SB, LaCl 3 and H 2 O 2 , at their optimum concentrations, improved the ascomycin yield by 33%, 30% and 26%, respectively, while GlcNAc and γ-butyrolactones, achieved only minor improvements at the same concentrations. [23].

Conclusions
Actinomycetes are considered as rich resources for natural products, with wide range bio-pharmaceutical applications and a rapidly rising number of genome sequencing information, which reveals their potential for SMs biosynthesis. Today, in the light of data obtained from the recently sequenced microbes as well as metagenomic libraries, scientists are aware of the value of untapped existing biosynthetic potential of SMs, yet only the upper tip of the iceberg has been scraped and many valuable natural products are still underexplored. In the same context, the discovery, as well as expansion, of antibiotics is considered as one of the top achievements in the field of curing and prevention of bacterial infections. Regrettably, new infectious diseases and resistant pathogens have been raised at an exceedingly high rate, with the absence of new discoveries of antibiotics that can keep up with this. It is speculated that the existing microbial reservoir needed for these discoveries is exhausted, especially with the increasing rates of antimicrobial resistance that immediately necessitate new approaches to stock-up such antimicrobial drug lines. Thus, it is highly significant to explore new biosynthetic pathways to drive the expression, progress and synthesis of new chemical scaffolds, especially since many of these pathways are observed to be unawake or rarely expressed. Awakening silent BGCs to produce plenty of SMs is considered difficult, but the careful organizing of the strategies described in our mini-review can now be much easier. The mentioned strategies in our mini-review have remarkably increased, especially over the past 5 years, due to genome sequencing and advances in elicitation tools that can make a conspicuous re-invigoration of natural product research. Apart from being potent antimicrobials, many of the chemically elicited SMs mentioned in this review exhibited significant potencies against diverse diseases, with the additional advantage of being pleiotropic regulators. Potent antitumor activities were observed with kigamicin D, mansouramycin, desferrioxamine B, desferrioxamine E, doxorubicin, baumycin, lankacidin, geldanamycin, surfactin lipopeptide 17 and actinomycin D. Actinomycin D, as a potent and clinically approved antitumor drug, also exhibited significant anti-HIV and anti-AIDs activities, similar to geldanamycin and the wide range of antibiotic primaricin, which followed the same behavior. On the other hand, pimaricin, candicidin, urauchimycin D and Acyl-surugamide A showed a remarkable antifungal efficacy, especially Acyl-surugamide A. Interestingly, dynactin, revealed a strong antiparasitic potential against peronosporomyctes, which sheds light on its effectiveness to be used as a potential and economic crop protector. Investigating the studied genera, most of the elicited SMs were detected from species belonging to Streptomyces, which represent about 75% of the studied genera, in addition to other less studied ones, all of which are illustrated in (Figure 11). Thus, the upcoming efforts for elicitation avenues should be directed towards the rarely studied, yet metabolites-rich, genera as Actinomyces, as well as other overlooked genera, as Nocardia, Pseudonocardia, Nocardiopsis, Salinispora and Rhodococcus. Among the studied techniques (Figure 12), the HiTES technique prevailed where the tested elicitors could enhance the largest portion of the reported SMs with 27.4 %, followed by antibiotics with 23.9 %, while the other groups of chemical elicitors apparently varied. The unique feature of bioactivity HiTES is the high throughput among hundreds of conditions that can be rapidly inspected for cryptic SMs induction. This can be greatly advantageous in directly linking the elicitor to the biosynthesis of the bioactive SM, and also permitting downstream mechanistic investigations that can address the base of the stimulatory activity. In addition, HiTES have recurrently highlighted antibiotics as inducers of silent BGCs, with stimulatory effects at SICs in contrast to inhibitory ones at higher doses. In the same context, atenolol (the antihypertension medication) had a completely different situation as it had no growthinhibitory effect against many Streptomyces species, such as S. hiroshimensis. On the other hand, it not only enhanced the production of taylorflavins, but also elicited the production of a variety of SMs, such as pyridindolol, hiroshidine and some other isocoumarins. In general, the deficient information about the precise physio-ecological signals makes the recent research on elicitation a really challenging mission. This requires interdisciplinary strategies that gather all the data collected from molecular biologists, microbiologists and natural product chemists. Efficient dereplication tools, such as metabolomics and molecular networking, are urgently needed to avoid isolation of previously isolated compounds, and to also appraise the possible novelty of the induced SMs. Furthermore, the structural elucidation of the new elicited SMs via different techniques, such as LC-MS, MS/MS and NMR, will directly lead to discovery of novel chemical scaffolds. Accordingly, future plans addressing the mechanism of induction will deliver additional insights into the regulatory elements that control the secondary metabolism. Apart from the technicalities of using different strategies for decryptification, it is also significant to understand why biosynthetic genes remain cryptic under normal laboratory conditions. Realizing the exact regulation of these cryptic systems would afford the possibility to explore the full potential of BGCs for the new SMs screening. It should be put into consideration that nature is not the only provider of novel natural products; chemical synthesis with modifications of existing moieties also offer potential alternatives. It is worth noting that more efforts should be directed towards exploring the marine environment that suggests novel and chemically rich species, but is underexplored in terms of elicitation. In general, the deficient information about the precise physio-ecological signals makes the recent research on elicitation a really challenging mission. This requires interdisciplinary strategies that gather all the data collected from molecular biologists, microbiologists and natural product chemists. Efficient dereplication tools, such as metabolomics and molecular networking, are urgently needed to avoid isolation of previously isolated compounds, and to also appraise the possible novelty of the induced SMs. Furthermore, the structural elucidation of the new elicited SMs via different techniques, such as LC-MS, MS/MS and NMR, will directly lead to discovery of novel chemical scaffolds. Accordingly, future plans addressing the mechanism of induction will deliver additional insights into the regulatory elements that control the secondary metabolism. Apart from the technicalities of using different strategies for decryptification, it is also significant to understand why biosynthetic genes remain cryptic under normal laboratory conditions. Realizing the exact regulation of these cryptic systems would afford the possibility to explore the full potential of BGCs for the new SMs screening. It should be put into consideration that nature is not the only provider of novel natural products; chemical synthesis with modifications of existing moieties also offer potential alternatives. It is worth noting that more efforts should be directed towards exploring the marine environment that suggests novel and chemically rich species, but is underexplored in terms of elicitation. We could overview the main chemical approaches available for unlocking the biosynthetic potential of Streptomycetes with a wide range, easily accessible, low-tech and low-cost methods from HiTES, to the more focused and complex strategies. It is clear that there is no single approach that can be dependably used to unlock every cryptic pathway. We have summarized the techniques that have been developed, to induce secondary metabolism by means of chemical approaches as well as recent advanced ones. We have highlighted current efforts to explore how secondary metabolism is controlled and, therefore, how the silent BGCs can be de-silenced for the aim of next-generation sequencing and advancement in the field of bioinformatics.
In conclusion, the exploration of under-discovered SMs from the veiled sources will ultimately announce solutions for the current crises of "slow moving drug development" and multi-drug-resistant pathogens. In reality, the consortia of such state-of-the-art strategies have reinforced the path for the discovery and development of novel chemical scaffolds to effectively fight multiple drug-resistant pathogens. A great burden faces the scientists now to conduct researches on the hidden features of the microbial treasure of SMs, for the exploration of yet-to be-identified lead bioactive compounds.